Ethenesulfonamide derivatives, a novel class of orally active endothelin-A receptor antagonists

Article abstract of DOI:10.1248/cpb.49.606

In the present article we wish to report the discovery of a novel class of ET_A-selective endothelin (ET), receptor antagonists through the modification of the ET_A/ET_B non-selective antagonist, Ro47-0203 (Bosentan, 1). Repl

Full text of DOI:10.1248/cpb.49.606

606
Chem. Pharm. Bull. 49(5) 606—612 (2001)
Vol. 49, No. 5
Ethenesulfonamide Derivatives, a Novel Class of Orally Active
Endothelin-A Receptor Antagonists
Hironori HARADA,* Jun-ichi KAZAMI, Susumu WATANUKI, Ryuji TSUZUKI,1) Katsumi SUDOH,
Akira FUJIMORI, Akihiro TANAKA,2) Shin-ichi TSUKAMOTO, and Isao YANAGISAWA
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305–8585,
Japan. Received January 11, 2001; accepted March 2, 2001
In the present article we wish to report the discovery of a novel class of ETA-selective endothelin (ET) recep-
tor antagonists through the modification of the ETA/ETB non-selective antagonist, Ro47-0203 (Bosentan, 1). Re-
placement of the benzenesulfonamide group of 1 with a 2-phenylethenesulfonamide group gave compound 5a
and resulted in improvement in ETA-selectivity. Optimization of the alkoxy side chain attached to the core pyrim-
idine ring yielded the 2-fluoroethoxy derivative (5n) with further improvement of ETA-selectivity. [IC50؍2.1 nM
for ETA receptor, ETB/ETA ratio؍1200]. After oral administration, compound 5n inhibited the big ET-1 induced
pressor response in pithed rats with a DR2 value of 2.6 mg/kg and also exhibited a potent antagonistic activity in
conscious rats.
Key words endothelin antagonist; ETA-selective; ethenesulfonamide
Endothelin (ET), isolated from a conditioned medium of benzenesulfonamide group by an alternative large hydropho-
cultured porcine vascular endothelial cells in 1988, is a bic group. We postulated that the vinylogous styryl group
highly potent vasoconstrictive 21-amino acid peptide.3) There could be designed as a surrogate for the phenyl group.13) Fur-
are three isoforms (ET-1, ET-2, ET-3). ET-1 is the predomi- thermore, since the SAR of the 6-position of the core pyrimi-
nant component of the three ET-isopeptides and derived from dine ring was not apparent at the beginning of our research,
precursor big ET-1.4) Largely because of its ability to con- we expected that judicious variations of the substituents of
strict vascular and nonvascular smooth muscle, ET-1 has this position could result in more ETA selective compounds.
been believed to be implicated in the pathogenesis of the var- Herein we report the discovery of a novel series of potent and
ious diseases such as myocardial infarction, hypertension, orally active nonpeptide ETA selective antagonists derived
heart failure, atherosclerosis, cerebral and coronary va- from 1.
sospasm, renal failure and asthma. In fact, it has been re-
Chemistry Charts 1 and 2 show the syntheses of 2-
ported that plasma ET-1 levels are increased in these phenylethenesulfonamide.
diseases5) and ET receptor antagonists have proven to be effi-
The starting compound (2) was prepared according to the
cacious in some animal models,6) suggesting that ET receptor method reported by Burri et al.11) Nucleophilic substitution
antagonists will be useful as a therapeutic agent.
of the pyrimidine derivative 2 with (E)-2-phenylethenesul-
Two subtypes of receptors for ETs, termed ETA receptor fonamide (3) resulted in the chloropyrimidine (4) in 88%
and ETB receptor, have been cloned and stably expressed in yield. The (E) form of 4 was confirmed by the coupling con-
1
mammals, and it appears that the ETA receptor exhibits affin- stant between vinyl protons (Jϭ19.5 Hz) in the H-NMR
ity for ET-1 and ET-2 over ET-3, whereas the ETB receptor spectrum. The chloropyrimidine 4 was treated with alkoxide
has nearly equipotent affinity for these three ETs.7)
in the corresponding alcohol by heating to give the hydrox-
Although a number of ETA-selective and ETA/ETB non-se- yalkoxy analogues (5a—c). When 4 was treated with 2-
lective non-peptide antagonists have been reported for a aminoethoxide in 2-aminoethanol by heating, the 2-hydrox-
decade,8) which type of antagonist would be better might de- yethylamino derivative (5d) was obtained instead of the 2-
pend on the target diseases. However, some literature sources aminoethoxy derivative. On the other hand, 4 and 2-
have indicated that the ETA-selective antagonists are the aminoethanol afforded the 2-aminoethoxy derivative (5e) at
promising candidates for the treatments of cardiovascular ambient temperature using sodium hydride. It is suggested
diseases such as hypertension and heart failure.9)
that the 2-hydroxyethylamino derivative 5d might be derived
We decided that our first goal was to discover ETA-selec- first by nucleophilic substitution of the chlorine group with
tive antagonists. At the beginning of our research, it seemed the alkoxide to give the 2-aminoethoxy derivative 5e, fol-
that Ro47-0203 (Bosentan) 1,8g) a potent, orally active and lowed by nucleophilic substitution of the alkoxy group with
ETA/ETB non-selective non-peptide antagonist , was an at- the amino group. In fact, heating the 2-aminoethoxy deriva-
tractive compound as a lead. Compound 1 possesses a central tive 5e in 2-aminoethanol at 80 °C afforded hydroxyethyl
pyrimidine nucleus bearing the pharmacophores indispens- amino derivative 5d.
able for activity and notably an acidic sulfonamide group ad-
Treatment of the amine 5e with acetylchloride gave the
jacent to the large hydrophobic 4-tert-butylphenyl group. acetamide (5f). Sulfonylation of the amine 5e with BOC-
While the acidic function in this area of the molecule seems NHSO2Cl followed by deprotection with trifluoro acetic acid
to be necessary to maintain activity,8,10) the role of the 4-tert- (TFA) gave the sulfamide (5g). Compound 5e was also
butylphenyl group in the binding to the receptor remains un- treated with the isopropylisocyanate to give the urea (5h)
certain.11,12)
(Chart 1).
First, we focused on the replacement of the 4-tert-butyl-
Nucleophilic substitution of the chloropyrimidine 4 with
To whom correspondence should be addressed. e-mail: haradah@yamanouchi.co.jp
© 2001 Pharmaceutical Society of Japan

May 2001
607
Reagents: BOCϭtert-butyloxycarbonyl, TFAϭtrifluoroacetic acid
Chart 1
Reagents: a) TFA. Trϭtriphenylmethyl
Chart 2
various alkoxides in DMF gave 5i, j and 5l—p in 20—72% to inhibit the ET-1 induced contraction of the ring prepara-
yield. The triphenylmethyl group in 5j was removed using tion sample of rat aorta. Some compounds were further ex-
TFA to give the imidazole (5k) in 42% yield. The carboxylic amined in vivo for their ability after intravenous or oral ad-
acid (5q) was generated by saponification of the correspond- ministration to inhibit an increase in mean arterial blood
ing ester 5i using 1 N NaOH in 63% yield. The ester 5i was pressure (MABP) due to the administration of exogenous big
treated with isopropylamine to give the amide (5r) in 46% ET-1 to pithed or conscious rats.
yield. Oxidation of the alcohol 5c using PDC gave the alde-
hyde (5s) in 59% yield (Chart 2).
The structure–activity relationships (SARs) of our novel
series of endothelin receptor antagonists are summarized in
The (E) form of all compounds was also confirmed by the Tables 1 and 2.
coupling constant between vinyl protons (JϾ15 Hz) in the
Replacement of the benzenesulfonamide group of 1 with
1H-NMR spectrum.
the 2-phenylethenesulfonamide group was investigated. The
2-phenylethenyl analogue 5a showed about 4-fold improve-
ment in binding affinity at the ETA receptor compared to 1
Results and Discussion
Compounds have been evaluated in vitro for their affinity and had an IC50 at the ETA receptor of 1.6 nM. This modifica-
for cloned human ETA and ETB receptors expressed in COS- tion also led to 6-fold increase in ETA selectivity over that of
1 cell by employing receptor-binding assays. Functional vas- the parent compound 1; the selectivity versus the ETB recep-
cular ET-1 antagonism was determined in vitro for the ability tor of 5a was found to be 230-fold. In order to confirm the

608
Vol. 49, No. 5
Table 1. ETA and ETB Receptor Binding Affinities for Ethenesulfonamide Table 3. Functional Vascular ET-1 Antagonism in Vitro; Inhibition of the
Derivatives ET-1 Induced Contraction of Ring Preparation Sample of Rat Aorta
Compound
pA2
na)
1
5a
5n
6.7
7.0
6.9
19
21
19
a) Values are the means of the indicated number of experiments (n).
IC50 (nM)a)
Selectivity
for ETA
Compound
R
c)
b)
b)
ETA
ETB
the core pyrimidine ring of 5a. There were few reports of
SARs around this side chain of 1 at the beginning of our re-
search.12)
1d)
6.6
1.6
250
370
38
230
90
5ae)
5b
–OCH2CH2OH
–OCH2CH2CH2OH
–OCH2CH2CH2CH2OH
–OCH2CH2NH2
11
990
Lengthening the distance between the alcohol oxygen and
the pyrimidine ring of 5a by one or two carbon units (5b, c)
resulted in decreases in ETA activity by 7-fold and 50-fold,
respectively. Replacement of the hydroxyl group in com-
pound 5a with an amino group led to a large loss of binding
affinity (5e). The imidazole derivative 5k and the acetic acid
derivative 5q were also less active than 5a, indicating that
basic or strong acidic groups were not well tolerated at this
position.
We further investigated the surrogates of the hydroxyl
group in compound 5a because of concern for the metabo-
lism in vivo of the alkyl alcohol moiety.14) We synthesized
the amide analogues 5r and 5f, the sulfonamide analogue 5l,
the sulfamide analogue 5g and the urea analogue 5h. These
derivatives have hydrogen bond donors in the side chain as
the hydroxyl analogue 5a. These were generally found to
have much lower binding affinity than 5a.
5ce)
5e
80
1000
13
100
5k
30
5q
5r
5f
–OCH2COOH
370
200
120
180
520
71
–OCH2CONHi-Pr
–OCH2CH2NHAc
–OCH2CH2NHMs
–OCH2CH2NHSO2NH2
–OCH2CH2NHCONHi-Pr
–NHCH2CH2OH
5l
5g
5h
5d
24
a) nϭ2 except 1 (nϭ7). b) Cloned human receptor binding. Blank space: not
tested. c) Expressed as ETB IC50/ETA IC50. d) Sodium salt. e) Pottasium salt.
Table 2. ETA and ETB Receptor Binding Affinities for Ethenesulfonamide
Derivatives
Replacement of the hydroxyethoxy group in 5a with the
hydroxyethylamino group gave 5d and resulted in reduced
activity by 15-fold. None of the investigated substituents
mentioned above resulted in improved ETA binding. These
SARs of the side chain led us to focus our attention on re-
placing the hydoroxyethoxy side chain in 5a with groups
bearing no hydrogen bond donor, but a hydrogen bond accep-
tor (Table 2).
IC50 (nM)a)
Selectivity
Compound
R
c)
for ETA
b)
b)
ETA
ETB
Although the ester analogue 5i and the aldehyde analogue
5s were significantly less active, the replacement of the hy-
droxyl group with a methoxy group (5m) was well tolerated
in ETA binding affinity and yielded the improvement in the
ETA selectivity. The replacement of the hydroxyl group with
a fluorine group (5n) led to a decrease in ETB affinity (from
370 to 2500 nM) with little change in ETA affinity (from 1.6
to 2.1 nM); the net result is a 5-fold improvement in ETA se-
lectivity. The ETA selectivity versus the ETB receptor was
found to be 1200-fold (5n), which was the best of the series.
1d)
5ae)
5i
6.6
1.6
100
130
4.8 2000
2.1 2500
120
280
250
370
38
230
–OCH2CH2OH
–OCH2CO2Et
–OCH2CH2CH2CHO
–OCH2CH2OMe
–OCH2CH2F
5se)
5m
5n
5o
417
1200
–OCH2CHF2
–OCH2CF3
5p
a—e) See footnotes in Table 1.
acidity of the sulfonamide moiety at the 4-position of the These results indicated that removal of a hydrogen bond
central pyrimidine ring, pKa values were measured for 1 and donor in the side chain lead to improved ETA selectivity.
5a. Compound 5a showed no significant difference in pKa
Compound 5n also blocked the contractions caused by ET-
value from 1 (5a, 4.7 vs. 1, 5.0). This result imply that the 1 in an isolated rat aorta in a concentration-dependent fash-
space of the phenyl group or the size of the 4-substituent of ion with pA2ϭ6.9 (Table 3). Further introduction of fluorine
the pyrimidine ring may participate in determining the affin- atoms to the side chain, affording 5o and 5p, caused reduced
ity for both ETA and ETB receptors.
binding affinity for the ETA receptor.
Compound 5a also blocked the contractions caused by ET-
Table 4 and Fig. 2 highlight our in vivo studies of the se-
1 in an isolated rat aorta in a concentration dependent fash- lected compounds.
ion with a pA2 value of 7.0. (pA2; see experimental section
and Table 3).
Compounds with a 2-phenylethenesulfonamide moiety
(5a, n) inhibited the increase in MABP due to the administra-
Intrigued by this observation, we then focused on the mod- tion of exogenous big ET-1 to anesthetized pithed rats after
ification of the hydroxyethoxy side chain at the 6-position of intravenous administration or oral administration (Table 4).

May 2001
609
ETA/ETB non-selective antagonist Ro47-0203 (Bosentan) 1
with 2-phenylethenylsulfonamide led to the discovery of a
new class of ETA selective endothelin antagonists. Among
this series, 5n (YM-62899) showed the highest ETA selectiv-
ity with potent affinity for ETA receptor. Compound 5n also
had good oral activity in the inhibition of the pressor re-
sponse caused by a big ET-1 infusion in both pithed and con-
scious rats. Compound 5n is a suitable tool for investigating
the therapeutic effects of an ETA selective antagonist in vari-
ous disease models. Further modification of 5n will be re-
ported elsewhere.
Fig. 1. Structure of R047-0203 (Bosentan, 1)
Experimental
Melting points were determined on a Yanagimoto micro melting point ap-
paratus without correction. 1H-NMR spectra were recorded on a JNM-
LA400, LA500, and A500 spectrometer using tetramethylsilane as an inter-
nal standard. MS spectra were determined with a Hitachi M-80 or JEOL
JMS-DX300 spectrometer. Elemental analysis data were withinϮ0.4% of
the calculated values unless otherwise noted. All organic extracts were dried
over anhydrous MgSO4. Chromatographic purification was performed on
Merck KGaA Silica gel 60 (0.040—0.063 mm).
(E)-N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-
4-yl]-2-phenylethenesulfonamide (4) To an ice-cooled solution of (E)-2-
phenylethenesulfonamide (3) (36.7 g, 200 mmol) in N,N-dimethylformamide
(DMF) (400 ml) was added 60% sodium hydride in mineral oil (17.6 g,
441 mmol), and the mixture was stirred for 30 min at room temperature. To
the mixture 4,6-dichloro-5-(2-methoxyphenoxy)pyrimidine (2) (70.0 g,
200 mmol) was added, and the mixture was stirred for 2 h at room tempera-
ture. It was poured into ice-water and 1 N HCl, and the resulting precipitate
was collected by filtration and washed with water and ethanol (EtOH) to
give 4 (92.2 g, 93%), 1H-NMR (DMSO-d6) d: 3.80 (3H, s), 6.81—6.91 (2H,
m), 7.06—7.16 (2H, m), 7.43—7.48 (3H, m), 7.70—7.80 (3H, m), 7.84—
7.94 (2H, m), 9.10 (2H, d, Jϭ6.0 Hz). FAB-MS m/z: 496 (Mϩϩ1). 1H-NMR
(DMSO-d6) at 90 °C d: 3.79 (3H, s), 6.84—6.90 (2H, m), 7.06—7.14 (2H,
m), 7.40—7.46 (3H, m), 7.62—7.68 (3H, m), 7.72 (1H, d, Jϭ19.5 Hz), 7.79
(1H, d, Jϭ19.5 Hz), 9.03 (2H, d, Jϭ6.0 Hz).
Fig. 2. Effect of p.o. Administration of the 2-Phenyethenesulfonamide De-
rivative 5n and 1 on Pressor Response to Big ET-1 in Conscious Normoten-
sive Rats
Change in MAP (%): Increase in mean arterial pressure (MAP) in conscious rats
elicited by intravenous administration of big ET-1 (0.5 nmol/kg). 1: Sodium salt.
Table 4. Effects of p.o. and i.v. Administration of the 2-Phenyethenesul-
fonamide Derivatives on Pressor Response to Big ET-1 in Pithed Rats
DR2 (mg/kg)a)
Compound
i.v.
nb)
p.o.
nb)
Potassium (E)-N-[6-(2-Hydroxyethoxy)-5-(2-methoxyphenoxy)-2-
(pyrimidin-2-yl)pyrimidin-4-yl]-2-phenylethenesulfonamidate (5a)
Sodium (230 mg, 10.0 mmol) was added to ethyleneglycol (5.6 ml,
100 mmol) and stirred at 60 °C until all the sodium was dissolved. (E)-N-
[6-chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]-2-
phenylethenesulfonamide (4) (495 mg, 1.00 mmol) was added to the solution
1
5a
5n
4.6
3.0
1.6
12
10
19
32
5.6
2.6
11
14
16
a) DR2 value was defined as the dose of tested compounds which was required to
produce a 2-fold rightward shift of dose–response curves of big ET-1 in diastolic blood
pressure (DBP). b) Values are the means of the indicated number of experiments (n).
and stirred at 80 °C for 3 h. It was poured into ice-water and 1
N HCl, and
the resulting precipitate was collected by filtration. The solid was chro-
matographed over silica gel using 20 : 1 CHCl3–methanol (MeOH) to give
(E )-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (500 mg, 96%), 1H-NMR
(DMSO-d6) d: 3.48—3.56 (2H, m), 3.83 (3H, s), 4.34—4.42 (2H, m), 4.68
In this study, the DR2 value was defined as the dose of the
test compound which was required to produce a 2-fold right- (1H, m), 6.72—6.88 (2H, m), 7.00—7.14 (2H, m), 7.38—7.50 (3H, m),
ward shift of the dose–response curves of big ET-1 in dias-
tolic blood pressure (DBP) (details are described in the ex-
7.62—7.78 (3H, m), 7.82 (1H, d, Jϭ15.5 Hz), 7.97 (1H, d, Jϭ15.5 Hz), 9.07
(2H, d, Jϭ4.5 Hz), 11.34 (1H, s). FAB-MS m/z: 522 (M+ϩ1). Anal. Calcd
for C25H23N5O6S: C, 57.57; H, 4.44; N, 13.43; S, 6.16. Found: C, 57.50; H,
4.40; N, 13.31; S, 6.13.
perimental section). Compounds 5a and 5n showed excellent
inhibitory activities after oral administration with DR2 values
of 5.6 and 2.6 mg/kg, respectively. The oral activity of com-
pound 5n was 12-fold more potent than that of 1. The calcu-
lated i.v./p.o. ratio from these DR2 values of 5n was 0.62.
Compounds 1 and 5n were tested for their antihyperten-
sive activity in conscious rats after oral administration. Com-
pound 5n also showed potent activity with a duration of ac-
tionϾ6 h (Fig. 2). Maximum inhibition of the pressor effect
of big ET-1 after oral administration was 83% for 1
(10 mg/kg) and 75% for 5n (3 mg/kg).
(E)-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (104 mg, 0.200 mmol) was
added to a 0.1 N potassium hydroxide (KOH) solution in EtOH (2 ml,
0.200 mmol) and stirred at room temperature for 30 min. The mixture was
concentrated in vacuo. The residue was crystallized from ether (Et2O) to
give 5a (84 mg, 75%). 1H-NMR (DMSO-d6) d: 3.52—3.60 (2H, m), 3.85
(3H, s), 4.26—4.36 (2H, m), 4.82—4.94 (1H, m), 6.40—6.52 (1H, m),
6.72—6.80 (1H, m), 6.84—6.92 (1H, m), 7.01 (1H, d, Jϭ8.0 Hz), 7.08—
7.20 (1H, m), 7.38—7.44 (3H, m), 7.54—7.68 (3H, m), 8.20 (1H, d,
Jϭ17 Hz), 9.03 (2H, d, Jϭ5.0 Hz). FAB-MS m/z: 560 (Mϩϩ1). Anal. Calcd
for C25H22N5O6SK·0.8H2O: C, 52.31; H, 4.14; N, 12.20; S, 5.59. Found: C,
52.50; H, 4.40; N, 12.08; S, 5.43.
These results indicated that our modification led to im-
provement in not only ETA selectivity in vitro but also oral
antagonistic activity in vivo.
In the same manner, compounds 5b and 5c were synthesized.
(E)-N-[6-(3-Hydroxypropoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (5b) 1.82 g (88%). mp
190—191 °C. 1H-NMR (DMSO-d6) d: 1.75—195 (2H, m), 3.54—3.60 (2H,
m), 3.98 (3H, s), 4.80 (2H, t, Jϭ5.5 Hz), 5.14—5.30 (1H, m), 6.89 (1H, t,
Jϭ7.5 Hz), 7.00 (1H, d, Jϭ7.0 Hz), 7.04—7.16 (2H, m), 7.32—7.42 (4H,
Conclusion
Replacement of the benzenesulfonamide group of m), 7.52—7.60 (2H, m), 7.62 (1H, d, Jϭ15.5 Hz), 8.04 (1H, d, Jϭ16 Hz),

610
Vol. 49, No. 5
8.81 (1H, s), 8.95 (2H, d, Jϭ4.5 Hz). FAB-MS m/z: 536 (Mϩϩ1). Anal.
Calcd for C26H25N5O6S·0.1Et2O: C, 58.40; H, 4.83; N, 12.90; S, 5.91.
Found: C, 58.44; H, 4.75; N, 12.61; S, 5.80.
CH2Cl2 (2 ml) was added trifluoroacetic acid (TFA) (2 ml), and the mixture
was stirred for 3 h at room temperature. The mixture was concentrated in
vacuo. The residue was chromatographed over silica gel using 20 : 1
CHCl3–MeOH to give an oil. The oil was crystallized from Et2O to give 5g
Potassium (E)-N-[6-(4-Hydroxybutoxy)-5-(2-methoxyphenoxy)-2-
(pyrimidin-2-yl) pyrimidin-4-yl]-2-phenylethenesulfonamidate Monohy-
drate (5c) 88 mg (72%). mp 141—142 °C. 1H-NMR (DMSO-d6) d:
1.20—1.36 (2H, m), 1.50—1.56 (2H, m), 3.20—3.30 (2H, m), 3.85 (3H, s),
4.25 (2H, t, Jϭ7.0 Hz), 4.31 (1H, t, Jϭ5.5 Hz), 6.35—6.50 (1H, m), 6.68—
6.80 (1H, m), 6.82—6.92 (1H, m), 7.01 (1H, d, Jϭ3.8 Hz), 7.06—7.20 (1H,
m), 7.28—7.48 (3H, m), 7.50—7.70 (3H, m), 8.20 (1H, d, Jϭ17 Hz), 9.02
(2H, d, Jϭ4.5 Hz). FAB-MS m/z: 550 (Mϩϩ1). Anal. Calcd for
C27H26N5O6SK·H2O: C, 53.54; H, 4.66; N, 11.56; S, 5.29; K, 6.45. Found:
C, 53.53; H, 4.63; N, 11.62; S, 5.30.
1
(140 mg, 51%), mp 192—194 °C. H-NMR (DMSO-d6) d: 3.04—3.14 (2H,
m), 3.83 (3H, s), 4.36—4.50 (2H, m), 6.62 (2H, s), 6.72—6.90 (2H, m),
7.00—7.12 (2H, m), 7.40—7.50 (3H, m), 7.66—7.78 (3H, m), 7.88 (1H, d,
Jϭ20 Hz), 7.98 (1H, d, Jϭ20 Hz), 9.08 (2H, d, Jϭ4.0 Hz), 11.43 (1H, s).
FAB-MS m/z: 600 (Mϩϩ1). Anal. Calcd for C25H25N7O7S·0.25H2O: C,
49.70; H, 4.25; N, 16.23; S, 10.62. Found: C, 49.55; H, 4.14; N, 16.24; S,
10.52.
(E)-N-{6-[2-(3-Isopropylureido)ethoxy]-5-(2-methoxyphenoxy)-2-
(pyrimidin-2-yl)pyrimidin-4-yl}-2-phenylethenesulfonamide (5h) To a
solution of 5e (400 mg, 0.768 mmol) in DMF (6 ml) was added 2-propyliso-
cyanate (78 mg, 0.922 mmol) in DMF, and the mixture was stirred for 3 h at
room temperature. The mixture was concentrated in vacuo. The residue was
chromatographed over silica gel using 20 : 1 CHCl3–MeOH to give an oil.
The oil was crystallized from Et2O–EtOH to give 5h (21 mg, 45%), mp
(E)-N-[6-(2-Hydroxyethylamino)-5-(2-methoxyphenoxy)-2-(pyrim-
idin-2-yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (5d) Sodium (69.3
mg, 30.2 mmol) was added to 2-aminoethanol (18.2 ml, 302 mmol) and
stirred at 60 °C until all the sodium was dissolved. Compound 4 (3.00 g,
6.03 mmol) was added to the solution and stirred at 60 °C for 1 h and 80 °C
for 2 h. It was poured into ice-water and 1 N HCl and neutralized with
NaHCO3. The mixture was then extracted with EtOAc. The organic layer
was washed with brine, dried, and concentrated in vacuo. The residue was
chromatographed over silica gel using 20 : 1 CHCl3–MeOH to give a solid.
The solid was washed with Et2O to give 5d (2.40 g, 76%). 1H-NMR
(DMSO-d6) d: 3.40—3.64 (4H, m), 3.85 (2.1H, s), 3.88 (0.9H, s), 4.74—
4.90 (1H, m), 6.72 (1H, t, Jϭ7.5 Hz), 6.87 (1H, d, Jϭ8.0 Hz), 6.94 (1H, t,
Jϭ7.5 Hz), 7.05 (1H, d, Jϭ8.0 Hz), 7.34—7.40 (2H, m), 7.40—7.60 (3H,
m), 7.60—7.74 (1H, m), 7.78—7.86 (0.7H, m), 8.00 (1H, d, Jϭ19 Hz), 9.02
(0.6H, d, Jϭ4.0 Hz), 9.15 (1.4H, d, Jϭ4.0 Hz), 10.74 (0.3H, s), 13.31 (0.7H,
s). FAB-MS m/z: 521 (Mϩϩ1). Anal. Calcd for C25H24N6O5S·0.3H2O: C,
57.09; H, 4.71; N, 15.98; S, 6.10. Found: C, 59.96; H, 4.44; N, 15.76; S, 6.10.
(E)-N-[6-(2-Aminoethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)-
pyrimidin-4-yl]-2-phenylethenesulfonamide (5e) To a solution of 2-
aminoethanol (1.23 g, 20.2 mmol) in DMF (10 ml) was added 60% sodium
hydride in mineral oil (1.13 g, 28.2 mmol), and the mixture was stirred for
30 min at room temperature. To the mixture, 4 (2.00 g, 4.03 mmol) was
added and the mixture was stirred for 1 h at room temperature. It was poured
into ice-water and neutralized with 1 N HCl. The mixture was then extracted
with CHCl3. The organic layer was washed with brine, dried, and concen-
trated in vacuo. The residue was crystallized from EtOH to give 5e (1.32 g,
63%), mp 187—189 °C. 1H-NMR (DMSO-d6) d: 3.80 (2H, t, Jϭ5 Hz), 3.84
(3H, s), 4.36—4.40 (2H, m), 6.48 (1H, d, Jϭ8.0 Hz), 6.73 (1H, t, Jϭ7.5 Hz),
6.89 (1H, t, Jϭ7.5 Hz), 7.01 (1H, d, Jϭ8.0 Hz), 7.18 (1H, d, Jϭ15 Hz), 7.31
(1H, t, Jϭ7.5 Hz), 7.36—7.42 (2H, m), 7.60 (2H, d, Jϭ7.5 Hz), 7.65 (1H, t,
Jϭ5.0 Hz), 8.21 (1H, d, Jϭ15 Hz), 9.04 (2H, d, Jϭ5.0 Hz). FAB-MS m/z:
521 (Mϩϩ1). Anal. Calcd for C25H24N6O5S·1.25H2O: C, 55.29; H, 4.92; N,
15.47; S, 5.90. Found: C, 55.02; H, 4.78; N, 15.60; S, 5.86.
(E)-N-[2-({5-(2-Methoxyphenoxy)-6-[(2-phenylethenesulfonyl)amino]-
2-(pyrimidin-2-yl)pyrimidin-4-yl}oxy)ethyl]acetamide (5f) To an ice-
cooled solution of 5e (275 mg, 0.528 mmol) in pyridine (5 ml) was added
acetylchloride (50 mg, 0.634 mmol), and the mixture was stirred for 2.5 h at
room temperature. The mixture was concentrated in vacuo. The residue was
chromatographed over silica gel using 20 : 1 CHCl3–MeOH to give an oil.
The oil was crystallized from Et2O to give 5f (180 mg, 61%), mp 186—
188 °C. 1H-NMR (DMSO-d6) d: 1.70 (3H, s), 3.16—3.28 (2H, m), 3.80 (3H,
s), 4.30—4.42 (2H, m), 6.74—6.88 (2H, m), 7.00—7.14 (2H, m), 7.38—
7.52 (3H, m), 7.60—7.74 (5H, m), 7.83 (1H, d, Jϭ15 Hz), 7.98 (1H, d,
Jϭ15 Hz), 9.08 (2H, d, Jϭ4.0 Hz), 11.40 (1H, s). FAB-MS m/z: 563
(Mϩϩ1). Anal. Calcd for C27H26N6O6S: C, 57.64; H, 4.66; N, 14.94; S, 5.70.
Found: C, 57.30; H, 4.67; N, 14.78; S, 5.75.
(E)-N-{5-(2-Methoxyphenoxy)-2-(pyrimidin-2-yl)-6-[2-(sulfamoy-
lamino)ethoxy]pyrimidin-4-yl}-2-phenylethenesulfonamide (5g) To an
ice-cooled solution of 5e (375 mg, 0.720 mmol) in pyridine (5 ml) was added
N-tert-butyloxycarbonylsulfamoylchloride (69.2 mg, 3.24 mmol) and the
mixture was stirred for 12 h at room temperature. The mixture was concen-
trated in vacuo. The residue was chromatographed over silica gel using 20 : 1
CHCl3–MeOH to give (E)-N-(6-{2-[(NЈ-tert-butoxycarbonylsulfamoyl)-
amino]ethoxy}-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl)-2-
phenylethenesulfonamide (342 mg, 68%). 1H-NMR (DMSO-d6) d: 1.29 (9H,
s), 3.10—3.28 (2H, m), 3.82 (3H, s), 4.37 (2H, t, Jϭ7.5 Hz), 6.70—6.86
(2H, m), 7.00—7.12 (2H, m), 7.36—8.04 (8H, m), 8.58 (1H, d, Jϭ5.5 Hz),
9.08 (2H, d, Jϭ6.0 Hz), 10.85 (1H, s), 11.56 (1H, s). FAB-MS m/z: 700
(Mϩϩ1). To an ice-cooled solution of (E)-N-(6-{2-[(NЈ-tert-butoxy-
carbonylsulfamoyl)amino]ethoxy}-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl)-2-phenylethenesulfonamide (320 mg, 0.457 mmol) in
1
177—179 °C. H-NMR (DMSO-d6) d: 0.98 (6H, d, Jϭ8.5 Hz), 3.04—3.22
(2H, m), 3.52—3.68 (1H, m), 3.82 (3H, s), 4.20—4.40 (2H, m), 5.58—5.82
(2H, m), 6.76—6.92 (2H, m), 7.00—7.16 (2H, m), 7.36—7.54 (3H, m),
7.62—7.80 (3H, m), 7.84 (1H, d, Jϭ20 Hz), 7.96 (1H, d, Jϭ20 Hz), 9.08
(2H, d, Jϭ4.0 Hz), 11.38 (1H, s). FAB-MS m/z: 606 (Mϩϩ1). Anal. Calcd
for C29H31N7O6S·0.25H2O: C, 57.08; H, 5.20; N, 16.07; S, 5.26. Found: C,
57.19; H, 5.07; N, 16.19; S, 5.34.
Ethyl (E)-({5-(2-Methoxyphenoxy)-6-[(2-phenylethenesulfonyl)amino]-
2-(pyrimidin-2-yl)pyrimidin-4-yl}oxy)acetate (5i) To an ice-cooled solu-
tion of ethyl 2-hydroxyacetate (2.10 g, 20.2 mmol) and 4 (2.00 g, 4.03 mmol)
in DMF (50 ml) was added 60% sodium hydride in mineral oil (1.14 g,
28.5 mmol), and the mixture was stirred for 2 h at room temperature and for
30 min at 50 °C. It was poured into ice-water and 1 N HCl, and the resulting
precipitate was collected by filtration. The solid was chromatographed over
silica gel using 20 : 1 CHCl3–MeOH to give an oil 5i (960 mg, 48%).
1H-NMR (DMSO-d6) d: 1.11 (3H, t, Jϭ6.5 Hz), 3.84 (3H, s), 4.08 (2H,
q, Jϭ6.5 Hz), 4.96 (2H, s), 6.72 (1H, d, Jϭ8.0 Hz), 6.82 (1H, t, Jϭ7.5
Hz), 7.04 (1H, t, Jϭ7.5 Hz), 7.10 (1H, d , Jϭ8.0 Hz), 7.40—7.48 (3H, m),
7.62—7.78 (3H, m), 7.83 (1H, d, Jϭ15.5 Hz), 7.93 (1H, d, Jϭ15.5 Hz), 9.05
(2H, d, Jϭ5.0 Hz), 11.53 (1H, s). FAB-MS m/z: 564 (Mϩϩ1). Anal. Calcd
for C27H25N5O7S·0.5H2O·0.4DMF: C, 56.28; H, 4.82; N, 12.57; S, 5.33.
Found: C, 56.44; H, 4.68; N, 12.70; S, 5.14.
(E)-N-[6-(1H-Imidazol-4-ylmethoxy)-5-(2-methoxyphenoxy)-2-
(pyrimidin-2-yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (5k) To a
solution of [1-(triphenylmethyl)imidazol-4-yl]methanol (673 mg, 1.98
mmol) in DMF (15 ml) was added 60% sodium hydride in mineral oil
(130 mg, 3.26 mmol). 4 (490 mg, 0.988 mmol) was added to the mixture and
the mixture was stirred for 3.5 h at 60 °C. It was poured into ice-water and
acidified with 1 N HCl (pHϭ4). The mixture was then extracted with EtOAc.
The organic layer was washed with brine, dried, and concentrated in vacuo.
The residue was chromatographed over silica gel using 40 : 1 CHCl3–MeOH
to give an oil. The oil was crystallized from Et2O–EtOH to give (E)-N-{5-(2-
methoxyphenoxy)-2-(pyrimidin-2-yl)-6-[1-(triphenylmethyl)imidazol-4-yl-
methoxy]-pyrimidin-4-yl}-2-phenylethenesulfonamide 5j (320 mg, 20%).
1H-NMR (DMSO-d6) d: 3.75 (3H, s), 5.29 (2H, s), 6.45 (1H, d, Jϭ7.5 Hz),
6.63 (1H, t, Jϭ7.5 Hz), 6.88—7.04 (7H, m), 7.06—7.34 (10H, m), 7.36—
7.40 (6H, m), 7.72—8.08 (4H, m), 8.81 (2H, d, Jϭ4.5 Hz). FAB-MS m/z:
800 (Mϩϩ1). A solution of 5j in TFA (10 ml) was stirred at room tempera-
ture for 30 min. The mixture was concentrated in vacuo. To the residue was
added sat.NaHCO3 and the resulting precipitate was collected by filtration.
The solid was chromatographed over silica gel using 20 : 1 CHCl3–MeOH to
give a solid. The solid was washed with Et2O to give 5k (225 mg, 42%), mp
1
147—149 °C. H-NMR (DMSO-d6) d: 3.79 (3H, s), 5.34 (2H, s), 6.59 (1H,
d, Jϭ8.0 Hz), 6.75 (1H, t, Jϭ8.0 Hz), 6.98 (1H, t, Jϭ8.0 Hz), 7.04 (1H, d,
Jϭ8.0 Hz), 7.28 (1H, s), 7.38—7.50 (3H, m), 7.63 (1H, d, Jϭ15.5 Hz),
7.64—7.80 (3H, m), 7.96—8.08 (2H, m), 9.15 (2H, d, Jϭ5.0 Hz). FAB-MS
m/z: 558 (Mϩϩ1). Anal. Calcd for C27H23N7O5S·1.5H2O: C, 55.47; H, 4.48;
N, 16.77; S, 5.49. Found: C, 55.35; H, 4.24; N, 16.76; S, 5.44.
(E)-N-[6-(2-Fluoroethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (5n) To an ice-cooled so-
lution of 2-fluoroethanol (258 mg, 4.03 mmol) in DMF (20 ml) was added
60% sodium hydride in mineral oil (194 mg, 484 mmol) and the mixture was
stirred at room temperature for 30 min. 4 (400 mg, 0.807 mmol) was added
to the solution and stirred at room temperature for 3 h. It was poured into
ice-water and 1 N HCl. The mixture was then extracted with CHCl3. The or-
ganic layer was concentrated in vacuo. The residue was chromatographed

May 2001
611
over silica gel using 40 : 1 CHCl3–MeOH to give an oil. The oil was crystal- idin-2-yl) pyrimidin-4-yl]-2-phenylethenesulfonamide (free acid of 5c)
lized from Et2O–EtOH to give 5n (240 mg, 57%). mp 142—143 °C. 1H- (1.55 g, 2.81 mmol) in dichloromethane was added pyridinium dichromate
NMR (DMSO-d6) d: 3.82 (3H, s), 4.40—4.68 (4H, m), 6.78 (1H, d, (PDC) (2.70 g, 7.17 mmol), and the mixture was stirred at room temperature
Jϭ8.0 Hz), 6.83 (1H, t, Jϭ7.5 Hz), 7.05 (1H, t, Jϭ7.5 Hz), 7.09 (1H, d,
overnight. To the mixture was added PDC (1.21 g, 3.21 mmol). After the
Jϭ8.0 Hz), 7.40—7.52 (3H, m), 7.62—7.80 (3H, m), 7.84 (1H, d, Jϭ mixture was stirred for 6 h, the mixture was filtered. The filtrate was concen-
16.0 Hz), 7.98 (1H, d, Jϭ16.0 Hz), 9.08 (2H, d, Jϭ4.0 Hz), 11.47 (1H, s). trated in vacuo. The residue was chromatographed over silica gel using 30 : 1
FAB-MS m/z: 524 (Mϩϩ1). Anal. Calcd for C25H22N5O5SF: C, 57.35; H, CHCl3–MeOH to give 905 mg (59%) of (E)-N-[6-(3-formylpropoxy)-5-(2-
4.24; N, 13.38; S, 6.12; F, 3.63. Found: C, 57.21; H, 4.34; N, 13.44; S, 6.09; methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl]-2-phenylethenesulfon-
F, 3.60.
In the same manner, compounds 5l, 5m, 5o, and 5p were synthesized.
amide as an amorphous solid. MS m/z: 548 (Mϩϩ1). (E)-N-[6-(3-formyl-
propoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]-2-
(E)-N-{6-[2-(Methanesulfonylamino)ethoxy]-5-(2-methoxyphenoxy)- phenylethenesulfonamide (104 mg, 0.200 mmol) was added to a 0.1 N KOH
2-(pyrimidin-2-yl)pyrimidin-4-yl}-2-phenylethenesulfonamide (5l) Yel- solution in EtOH (1.75 ml, 0.175 mmol) and stirred at room temperature for
low amorphous (420 mg, 69%). 1H-NMR (DMSO-d6) d: 2.83 (3H, s), 30 min. The mixture was concentrated in vacuo. The residue was crystal-
3.14—3.26 (2H, m), 3.83 (3H, s), 4.34—4.50 (2H, m), 6.74—6.98 (2H, m),
lized from Et2O to give 5s (88 mg, 72%). mp 140—145 °C. 1H-NMR
7.00—7.16 (2H, m), 7.27 (1H, t, Jϭ6.0 Hz), 7.38—7.52 (3H, m), 7.62— (DMSO-d6) d: 1.30—1.48 (1H, m), 1.50—1.60 (1H, m), 1.70—1.82 (1H,
7.80 (3H, m), 7.85 (1H, d, Jϭ15.0 Hz), 7.97 (1H, d, Jϭ15.0 Hz), 9.08 (2H, m), 2.22—2.32 (1H, m), 3.80 (3H, s), 4.18—4.30 (2H, m), 6.39—6.52 (1H,
d, Jϭ4.0 Hz), 11.44 (1H, s). FAB-MS m/z: 599 (Mϩϩ1). Anal. Calcd for m), 6.69—6.78 (1H, m), 6.80—6.94 (1H, m), 6.98—7.06 (1H, m), 7.08—
C26H26N6O7S2·0.5H2O·1.7CHCl3: C, 50.06; H, 4.36; N, 13.38; S, 10.21; Cl,
2.88. Found: C, 50.20; H, 4.37; N, 13.55; S, 9.92; Cl, 2.62.
(E)-N-[6-(2-Methoxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
7.20 (1H, m), 7.28—7.36 (1H, m), 7.38—7.46 (2H, m), 7.56—7.66 (3H, m),
8.24 (1H, d, Jϭ15 Hz), 9.02 (2H, d, Jϭ5.0 Hz), 9.51 (1H, s). FAB-MS m/z:
548 (Mϩϩ1). Anal. Calcd for C27H24N5O6SK·H2O: C, 53.72; H, 4.34; N,
yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (5m) Crystallized from 11.60; S, 5.31; K, 6.48. Found: C, 53.61; H, 4.47; N, 11.41; S, 5.25.
1
Et2O (240 mg, 56%), mp 129—130 °C. H-NMR (DMSO-d6) d: 3.08 (3H,
s), 3.36—3.48 (2H, m), 3.82 (3H, s), 4.42—4.56 (2H, m), 6.76—6.90 (2H, to each membrane preparation, which was incubated with various concentra-
Binding Assay For competition studies, [125I]ET-1 (200 pM) was added
m), 7.00—7.14 (2H, m), 7.40—7.54 (3H, m), 7.62—7.80 (3H, m), 7.82 (1H,
d, Jϭ16.0 Hz), 7.97 (1H, d, Jϭ16.0 Hz), 9.00—9.14 (2H, m), 11.41 (1H, s).
FAB-MS m/z: 536 (Mϩϩ1). Anal. Calcd for C26H25N5O6S: C, 58.31; H,
4.70; N, 13.08; S, 5.99. Found: C, 58.26; H, 4.83; N, 13.10; S, 5.96.
tions of compounds in 250 ml of assay buffer containing 50 mM Tris–HCl,
pH 7.4, 10 mM MgCl2 and 0.01% BSA. Binding reactions were initiated by
the addition of the membrane preparations. After the incubation period
(180 min, room temperature), the reaction was terminated by the addition of
3 ml of ice-cold Tris buffer (50 mM Tris–HCl, pH 7.4, 10 mM MgCl2 and
(E)-N-[6-(2,2-Difluoroethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (5o) Crystallized from 0.01% BSA) followed by rapid filtration through Whatman GF/C filters. The
1
Et2O (246 mg, 58%). mp 190—191 °C. H-NMR (DMSO-d6) d: 3.81 (3H, filters were rinsed twice, and the radioactivity retained on the filters was
s), 4.66 (2H, t, Jϭ15.0 Hz), 6.15 (1H, t, Jϭ55 Hz), 6.78—6.85 (2H, m), counted using a gamma counter at 60% efficiency. Each assay was per-
7.04—7.10 (2H, m), 7.45—7.46 (3H, m), 7.70—7.74 (3H, m), 7.86 (1H, d, formed in duplicate, and nonspecific binding was assessed in the presence of
Jϭ15.0 Hz), 7.98 (1H, d, Jϭ15.0 Hz), 9.10 (2H, d, Jϭ5.0 Hz), 11.57 (1H, s).
FAB-MS m/z: 542 (Mϩϩ1). Anal. Calcd for C25H21N5O5SF2: C, 55.45; H,
3.91; N, 12.93; S, 5.92; F, 7.02. Found: C, 55.46; H, 3.82; N, 12.95; S, 5.95;
F, 6.98.
100 nM unlabeled ET-1. The IC50 values were calculated by a non-linear re-
gression analysis.
Vessel Contraction (Inhibition of Big ET-1 Induced Contraction of
Ring Preparation Sample of Rat Aorta) Male Wistar rats were anes-
thetized with sodium pentobarbital (60 mg/kg i.p.) and the thoracic aorta
was quickly removed and placed in a Krebs–Henseleit solution (118.4 mM
NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, 2.5 mM
CaCl2, 11.1 mM glucose). The endothelium was removed by gentle rubbing
(E)-N-[5-(2-Methoxyphenoxy)-2-(pyrimidin-2-yl)-6-(2,2,2-trifluo-
roethoxy)pyrimidin-4-yl]-2-phenylethenesulfonamide (5p) Crystallized
1
from Et2O (325 mg, 72%), mp 204—205 °C. H-NMR (DMSO-d6) d: 3.79
(3H, s), 5.03 (2H, q, Jϭ8.5 Hz), 6.76—6.88 (2H, m), 7.02—7.14 (2H, m),
7.42—7.52 (3H, m), 7.66—7.80 (3H, m), 7.87 (1H, d, Jϭ15.0 Hz), 7.96 of the intimal surface using a small cotton ball and each ring was suspended
(1H, d, Jϭ15.0 Hz), 9.09 (2H, d, Jϭ4.5 Hz), 11.67 (1H, s). AB-MS m/z: 560 in a 10 ml isolated organ chamber (siliconized) containing gassed (95%
(Mϩϩ1). Anal. Calcd for C25H20N5O5SF3: C, 53.67; H, 3.60; N, 12.52; S, O2–5% CO2) and warmed (37 °C) Krebs–Henseleit solution. Vessel seg-
5.73; F, 10.19. Found: C, 53.53; H, 3.60; N, 12.43; S, 5.68; F, 10.28.
(E)-({5-(2-Methoxyphenoxy)-6-[(2-phenylethenesulfonyl)amino]-2-
ments were attached to an isometric force transducer linked to a physio-
graphic recorder for monitoring tension change. Baseline tension was set at
(pyrimidin-2-yl)pyrimidin-4-yl}oxy)acetic Acid (5q) To a solution of 5i 1.0 g, and the tissues were allowed to equilibrate for 60 min. The tissues
(490 mg, 0.869 mmol) in THF (10 ml) was added 1 N sodium hydroxide in were contracted with phenylephrine (1 mM) followed by challenge with
water (3.48 ml, 3.28 mmol) and the mixture was stirred for 12 h at room tem- acetylcholine (1 mM). A negative relaxant response to acetylcholine con-
perature. The mixture was concentrated in vacuo. To the residue 1 N HCl in firmed the absence of endothelium. The rings were stimulated to contract
water was added and the resulting precipitate was collected by filtration. The with 60 mM KCl repeatedly until the contractile response to KCl became sta-
solid was washed with water and EtOH to give 5q (294 mg, 63%), mp 206— ble before starting the experiments. Cumulative concentration–response
207 °C. 1H-NMR (DMSO-d6) d: 3.84 (3H, s), 4.92 (2H, s), 6.74 (1H, d, curves to ET-1 were performed in the presence or absence of test compounds
Jϭ8.0 Hz), 6.79—6.81 (1H, m), 7.01—7.04 (1H, m), 7.09 (1H, d,
Jϭ8.0 Hz), 7.40—7.52 (3H, m), 7.64—7.78 (3H, m), 7.82 (1H, d, Jϭ
after a 30-min pretreatment period. Contractile responses were expressed as
a percentage of the response elicited by 60 mM KCl. The effective concentra-
15.0 Hz), 7.95 (1H, d, Jϭ15.0 Hz), 9.06 (2H, d, Jϭ4.5 Hz), 11.47 (1H, s). tion of ET-1 causing 50% maximum response (EC50) in the presence or ab-
AB-MS m/z: 536 (Mϩϩ1). Anal. Calcd for C25H21N5O7S·0.25H2O: C, sence of test compounds was determined by regression analysis. Dose ratios
55.60; H, 4.01; N, 12.97; S, 5.94. Found: C, 55.54; H, 4.13; N, 12.71; S, were determined and the results analyzed for competitiveness. The pA2 value
5.83.
(E)-N-Isopropyl-2-({5-(2-methoxyphenoxy)-6-[(2-phenylethenesul-
fonyl)amino]-2-(pyrimidin-2-yl)pyrimidin-4-yl}oxy)acetamide (5r)
was estimated by plotting the log of (dose ratioϪ1) as a function of the neg-
ative log of concentration of test compounds.
Functional Assay in Vivo (Inhibition of Pressor Response to Big ET-
Compound 5i (300 mg, 0.532 mmol) and isopropylamine (3 ml) were heated 1): Pithed Rats In vivo antagonistic activity in pithed rats was evaluated
under reflux for 5.5 h. The mixture was concentrated in vacuo. The residue according to the method of Clozel et al. described previously (Clozel et al.,
was chromatographed over silica gel using 20 : 1 CHCl3–MeOH to give an 1994). Briefly, male Wistar rats were pithed under sodium pentobarbital
amorphous solid. The amorphous solid was crystallized from Et2O to give
5r (140 mg, 46%), mp 149—150 °C. H-NMR (DMSO-d6) d: 0.96 (6H, d, carotid artery and the left femoral vein were cannulated for blood pressure
Jϭ8.5 Hz), 3.72—3.84 (1H, m), 3.84 (3H, s), 4.76 (2H, s), 6.76—6.90 (2H, measurements and i.v. injection of drugs, respectively. After stabilization of
anesthesia and artificially ventilated with room air. The right common
1
m), 6.98—7.16 (2H, m), 7.24—7.36 (1H, m), 7.40—7.54 (3H, m), 7.62—
blood pressure, various doses of (1 ml/kg) test compounds or vehicle (dis-
7.78 (3H, m), 7.83 (1H, d, Jϭ20 Hz), 7.94 (1H, d, Jϭ20 Hz), 9.06 (2H, d, tilled water) were injected. Five minutes later, the first dose of big ET-1 was
Jϭ5.5 Hz), 11.48 (1H, s). FAB-MS m/z: 577 (Mϩϩ1). Anal. Calcd for injected intravenously. In another series of experiments, the oral activities of
C28H28N6O6S·H2O: C, 56.56; H, 5.08; N, 14.13; S, 5.39. Found: C, 56.67;
H, 4.96; N, 14.17; S, 5.56.
test compounds were assessed. Varying doses of (5 ml/kg) the test com-
pounds or vehicle (0.5% methyl cellulose) were administered by gastric gav-
Potassium (E)-N-[6-(3-formylpropoxy)-5-(2-methoxyphenoxy)-2- age with a cannula. About 20 minutes later, the rats were anesthetized with
(pyrimidin-2-yl) pyrimidin-4-yl]-2-phenylethenesulfonamidate (5s) To sodium pentobarbital, and 30 minutes later, pithed and ventilated. After sta-
a solution of (E)-N-[6-(4-hydroxybutoxy)-5-(2-methoxyphenoxy)-2-(pyrim- bilization of the blood pressure, the first dose of big ET-1 was injected intra-

612
Vol. 49, No. 5
venously. In this study, the DR2 value was defined as the dose of test com-
pounds which was required to produce a 2-fold rightward shift of the dose–
response curves of big ET-1 in diastolic blood pressure (DBP).
Webb M. L., Zhang R., Hunt J. T., J. Med. Chem., 38, 1344 (1995); b)
Doherty A. M., Patt W. C., Edmunds J. J., Berryman K. A., Reisdorph
B. S., Plummer M. S., Shahripour A., Lee C., Cheng X.-M., Walker D.
M., Haleen S. J., Keiser J. A., Welch K. M., Hallak H., Taylor D. G.,
Reynolds E. E., ibid., 38, 1259—1263 (1995); c) Roux S., Breu V.,
Giller T., Neidhart W., Ramuz H., Coassolo P., Clozel J. P., Clozel M.,
J. Pharmacol. Exp. Ther., 283, 1110—1118 (1997); d) Wu C., Chan
M. F., Stavros F., Raju B., Okun I., Mong S., Keller K. M., Brock T.,
Kogan T. P., Dixon R. A. F., J. Med. Chem., 40, 1690—1697 (1997); e)
Winn M., von Geldern T. W., Opgenorth T. J., Jae H.-S., Tasker A. S.,
Boyd S. A., Kester J. A., Mantei R. A., Bal R., Sorensen B. K., Wu-
Wong J. R., Chiou W. J., Dixon D. B., Novosad E. I., Hernandes L.,
Marsh K. C., ibid., 39, 1039 (1996); f ) Riechers H., Albrecht H.-P.,
Amberg W., Baumann E., Bernard H., Bohm H.-J., Klinge D., Kling
A., Muller S., Raschak M., Unger L., Walker N., Wernet W.,. ibid., 39,
2123 (1996); g) Clozel M., Breu V., Gray G. A., Kalina B., Loffler B.
M., Burri K., Cassal J. M., Hirth G., Muuler M., Neidhart W., Ramuz
H., J. Pharmacol. Exp. Ther., 270, 228—235 (1994); h) Elliott J. D.,
Lago M. A., Cousins R. D., Gao A., Leber J. D., Erhard K. F., Nambi
P., Elshourbagy N. A., Kumar C., Lee J. A., Bean J. W., DeBrosse C.
W., Eggleston D. S., Brooks D. P., Fueurstein G., Ruffolo R. R., Wein-
stock J., Gleason J. G., Peishoff C. E., Ohlstein E. H., J. Med. Chem.,
37, 1553—1557 (1994).
Functional Assay in Vivo (Inhibition of Pressor Response to Big ET-
1): Conscious Normotensive Rats Male Wistar rats were anesthetized
with sodium pentobarbital (60 mg/kg i.p.). The right common carotid artery
and the left jugular vein were cannulated with a polyethylene tube for deter-
mination of blood pressure and heart rate, and for i.v. administration of big
ET-1 (0.5 nmol/kg). The animals were allowed to recover for 2 to 3 d after
the operation, during which time they were housed in individual cages with
free access to rat chow and water. After an appropriate equilibration period,
bolus i.v. doses of big ET-1 were administered to determine control re-
sponses and patency of the catheters. Each rat was treated with a single p.o.
dose of antagonist or vehicle (0.5% methyl cellulose) and any changes in
blood pressure were noted. The percentage of the pressor response to big
ET-1 challenges during the subsequent 6.5 h and at 24 h were used as a mea-
sure of big ET-1 inhibition.
Acknowledgment We are grateful to Dr. Toshio Okazaki and Dr.
Shuichi Sakamoto for their advice. We thank Mr. Masanao Sanagi and Miss
Akiko Koakutsu for pharmacological study. We also thank members of the
Division of Analytical Research for performing instrumental analyses.
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